The overall objective of this project is to define and characterize normal stem cell biology with particular emphasis on the hemopoietic stem cell. The reseach is focused on the following areas: (1) employ current stem cell assays to identify the growth regulators which act upon these primitive cells; (2) continue to devise novel methods for both maintaining the stem cells in culture for longer periods of time and to improve the recognition of stem cells as well as further defining their progeny; and (3) purification, preparation of a panel of monoclonal antibodies and molecular cloning of erythropoietin . We previously reported that a single cell line derived from a mouse with erythroleukemia produced factors with erythropoietic activity. We have recently found that other lines, isolated several years ago, also produced erythropoietin. Extensive evidence indicate that erythropoietic activity produced by these lines is mouse erythropoietin. Our characterization of the erythropoietic activity included (A) biological (active in nine biological assays), (B) immunological (four anti-erythropoietin sera), and (C) biochemical (enzyme treatments and multiple purification techniques) studies. The finding that a large percentage of cells became benzidine- and spectrin-positive upon hemin induction indicated that most of the cells wer erythroid and suggested autocrine production of erythropoietin. We therefore tested cloned lines for heterogeneity with respect to erythropoietin production. Since erythropoietin was detected in media from each of the 20 lines derived by 2 cycles of single cell cloning, it is likely that, in these lines at least, the same cells which produce erythropoietin also have erythroid developmental potential. Conditions have been found which allow production of more than 1 U/ml erythropoietin in cultures that are free of serum and albumin.